The clonal selection theory of Burnet, which explains the general basis of antibody production, has gained virtually complete acceptance. Burnet, M. (1961) Sci. Am. 204:58; Jerne, N. K. (1976) Harvey Lecture 70:93. The theory is based on several premises: (1) as individual cells, i.e., lymphocytes, in the immune system differentiate, each becomes capable of producing only one species of antibody molecule; (2) the entire spectrum of possible antibody-producing cells is present within the lymphoid tissues prior to stimulation by any antigen; that is, the step in which each lymphocyte becomes specified to produce only one type of antibody molecule occurs in the absence of a potential antigen for that antibody; and (3) lymphocytes capable of producing an antibody specific to a particular antigen are induced, by the presence of that antigen, to proliferate and to produce large quantities of the antibody. An enormous range of genetically unique lymphoid cells is present in the lymphoid organs, e.g., the spleen, of each mammal. The spleen can be considered a library of cells, each of which can manufacture a unique antibody, and the library is so large that for any particular antigen, at least one lymph cell exists within the library that is capable of recognizing the antigen and producing antibodies specific to the antigen.
Heretofore, the production of an antibody that will recognize an antigen of interest has required the antigenic stimulation of a laboratory animal. Typically, the antigen is injected into a laboratory animal, and, after a suitable incubation period, a second injection is given. The spleen cells of the animal are then harvested and fused to myeloma cells. When fused to a spleen cell, the myeloma cell confers to the spleen cell its ability to grow in culture. Surviving colonies of fused cells, i.e., hybridomas, are then screened to identify clones that produce antibodies that specifically recognize the antigen. This procedure must be repeated each time it is desired to produce an antibody to a particular antigen. For each antigen of interest, it is necessary to (1) antigenically stimulate an animal, (2) remove its spleen and hybridize the spleen cells with myeloma cells, and (3) dilute, culture, and screen clones for specific antibody production. Though antibodies that recognize the antigen are produced, this technique does not identify the epitope, i.e., the specific site on the antigen that an antibody recognizes; and one cannot direct the development of antibodies specific to a particular predetermined site or region of the antigen. Also, hybridoma techniques are not effective in the direct development of monoclonal antibodies that recognize haptens, i.e., molecules that contain constitute antibody recognition sites, but which do not elicit an antigenic reaction when injected without a carrier into a laboratory animal. Since antigenic stimulation and antibody production are potentially hazardous to the host, the use of human hosts has been precluded in the development of monoclonal antibodies.
The universe of antibody binding specificities may be open or closed. If the universe of antibody binding specificities is closed, then the following basic tenets apply:
a) one can design and prepare any given epitope and isolate any antibody (for example, a monoclonal antibody produced by a member of a random set of hybridomas) from a universe of antibodies without having first immunized an experimental animal with an antigen containing that epitope. A self-addressing sorting scheme can be used to screen to identify the proper paired correspondence between antibody and epitope; PA1 b) the universe of epitopes can be specified in at least a theoretical fashion, and in principle, can be synthesized; and PA1 c) one can independently isolate and identify an antibody-producing hybridoma with the same epitopic specificity as one previously isolated and identified. Such a repeated isolation occurs in a "second hit" experiment, and can be used to estimate the effective size of the universe of antibody specificities. Such an approach is similar in logic to defining a complementation group in genetics. PA1 a) one cannot isolate an antibody specific for an epitope without prior immunization with an antigen containing that epitope; PA1 b) the universe of epitopes cannot be specified or synthesized; and PA1 c) one should not be able to independently isolate more than one antibody with the same target specificity.
Even if the universe of epitopes is large, if it is closed, it can be defined by rules, algorithms or iterative analyses.
In the alternative, if the universe of antibody specificities is open, the following principles apply:
The binding domain of a monoclonal antibody specific to a malaria virus surface protein has been identified as being no larger than 40 amino acids long. Cochrane, A. H. et al. Proc. Natl. Acad. Sci. U.S.A. 79:5651 (1982), inserted a 340 base pair sequence from a Plasmodium knowlesi gene into the pBR322 vector. The engineered vector produced in E. coli a beta-lactamase fusion polypeptide that reacted with a monoclonal antibody specific for a P. knowlesi circumsporozooite (CS) protein. This finding indicated that the binding domain of the monoclonal antibody was limited to a region of the CS protein encoded by the inserted sequence, or approximately 110 amino acids. Lupski, J. R. et al., Science 220:1285 (1983), used the same system and, employing transposition mapping techniques, further localized the binding domain to a 40-amino acid region of the CS protein.
Green, N. et al., published PCT application 84/00687, produced antibodies by inoculating laboratory animals with synthetic peptides. Antibodies produced in response to peptides having a length of 8 to 40 amino acid residues and corresponding to sequences in an influenza virus protein were cross-reactive with the virus in vitro.
Dame, J. B. et al., Science 225:593 (1984), sequenced the CS gene of Plasmodium falciparum and discovered 41 tandem repeats of a tetrapeptide, with some minor variations. Using synthetic peptides of 4, 7, 11, and 15 amino acid residues of the predominant repeating amino acid sequence, Dame et al. then conducted competitive binding assays to determine what length of peptide would inhibit the binding of the CS protein with a monoclonal antibody specific to that protein. Dame et al. found that the synthetic 4 amino acid sequence did not significantly inhibit binding, but the 7, 11 and 15 amino acid sequences did inhibit binding. These results suggest that this monoclonal antibody to the CS protein recognizes a 5 to 7 amino acid sequence comprising the repeating tetrapeptide.
The known crystal structures of the Fab fragment and lysozyme show that there are two contact points on the lysozyme molecule for the antibody combining site, and each contact point spans over about five amino acids. Earlier work on antibody binding to carbohydrate antigens and glycosidase cleavage protection experiments show that 5-6 sugar residues are protected from glycosidase cleavage. Studies with antibody binding to haptens also suggests that antibody sites are small. Peptide competition experiments, also called epitope mapping experiments, show that oligopeptides 4 to 5 amino acids in length can specifically compete for antibody binding.
In addition, linear sequences which differ in only one amino acid, can compete for antibody binding with varying degrees of specificity (see, e.g., Geysen et al. (1986) in Synthetic Peptides as Antigens; Ciba Foundation Symposium 119, R. Porter and J. Wheelan, Eds. (New York, Wiley) pp. 130-149).
While five amino acids is a representative length of peptide sequence which can bind with differential specificity to an antibody, five amino acid residues is not necessarily the size of an immunogenic peptide. Generally, when an oligopeptide is the desired immunogen, it is first conjugated to a larger carrier molecule. The actual operational relationship between the immunizing entity and the binding entity can only be resolved when an in vitro immunization-dependent antibody synthesis system is developed.